视黄酸诱导的小鼠神经管畸形基因表达趋势分析

doi:10.3969/j.issn.1674-8115.2024.07.007

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (7): 859-870.doi: 10.3969/j.issn.1674-8115.2024.07.007

• 论著 · 基础研究 • 上一篇

视黄酸诱导的小鼠神经管畸形基因表达趋势分析

曹睿¹^,²(), 卫凯鑫³, 张晓娜³, 刘雨榕³, 张丽³^,⁴()

^1.山西医科大学出生缺陷与细胞再生山西省重点实验室，太原 030001
^2.山西医科大学转化医学研究中心，太原 030001
^3.山西医科大学基础医学院生物化学与分子生物学系，太原 030001
^4.山西医科大学第一医院肝胆胰外科及肝移植中心，太原 030001

收稿日期:2024-01-06 接受日期:2024-03-25 出版日期:2024-07-28 发布日期:2024-07-28
通讯作者: 张丽 E-mail:caorui@d.sxmu.edu.cn;zhangli3788@163.com
作者简介:曹　睿（1984—），女，博士；电子信箱：caorui@d.sxmu.edu.cn。
基金资助:
国家自然科学基金青年项目(82201319);山西省基础研究计划面上项目(20210302123347)

Trend analysis of differentially expressed genes in retinoic acid-induced neural tube defects in mouse model

CAO Rui¹^,²(), WEI Kaixin³, ZHANG Xiaona³, LIU Yurong³, ZHANG Li³^,⁴()

^1.Shanxi Key Laboratory of Birth Defects and Cell Regeneration, Shanxi Medical University, Taiyuan 030001, China
^2.Translational Medicine Research Centre, Shanxi Medical University, Taiyuan 030001, China
^3.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, China
^4.Department of Hepatobiliary and Pancreatic Surgery and Liver Transplantation Centre, The First Hospital of Shanxi Medical University, Taiyuan 030001, China

Received:2024-01-06 Accepted:2024-03-25 Online:2024-07-28 Published:2024-07-28
Contact: ZHANG Li E-mail:caorui@d.sxmu.edu.cn;zhangli3788@163.com
Supported by:
National Natural Science Foundation of China(82201319);Basic Research Programme of Shanxi Province(20210302123347)

摘要/Abstract

摘要：

目的·探究小鼠胚胎在视黄酸（retinoic acid，RA）诱导下产生神经管畸形的分子调控机制，揭示小鼠神经管闭合阶段基因表达规律。方法·基于已获得的小鼠胚胎神经管闭合关键期［胚胎发育第8.5日（embryonic day 8.5，E8.5）、E9.5、E10.5］高质量脑泡转录组数据，利用短时间序列表达挖掘器（Short Time-series Expression Miner，STEM）软件分别得到RA处理组和正常组在3个时间点的基因表达趋势数据。对处理组与正常组基因表达趋势不一致的基因进行基因本体（Gene Ontology，GO）富集分析、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析，并随机筛选候选基因以验证测序数据可靠性。利用RA诱导构建神经管畸形小鼠模型，分为处理组和正常组，每组各9只。处理组和正常组孕鼠在E7.5分别接受28 mg/kg RA和香油灌胃处理，在E8.5、E9.5、E10.5收集胎鼠脑泡组织，对筛选的候选基因进行实时荧光定量PCR（quantitative real-time PCR，RT-qPCR）验证。结果·正常组共检测出18 255个基因的表达量数据，处理组共检测出19 037个基因的表达量数据；正常组基因可归纳至7个具有显著意义的表达模式中，处理组基因可归纳至6个具有显著意义的表达模式中；正常组和处理组检测到表达的基因数目足够、表达的模式相似，具有可比性。进一步分析发现正常组中呈现上升表达趋势但在处理组中呈现下降表达趋势的基因共有46个，在生物学过程层面富集在器官发育、神经元凋亡的正负调控、少突胶质细胞增殖、成纤维生长因子信号通路等；在细胞组分层面，主要参与组成细胞、神经元的基本结构；在分子功能层面，主要与成纤维细胞生长因子受体结合有关。正常组中呈现下降表达趋势而在处理组中呈现上升表达趋势的61个基因，在生物学过程层面富集在细胞溶解、氨基酸/离子转运等功能上；在细胞组分层面，富集在胞内分子、皮质颗粒、胞外区域、细胞间隙等；在分子功能层面，与一系列酶及转运蛋白的活性有关。RT-qPCR验证结果显示转录组测序数据真实可靠。结论·RA干预使小鼠胚胎发育过程中发生基因表达失调和应激反应，导致胚胎发育异常，机体自我保护相关信号通路激活，维持胚胎正常发育的基因受到抑制。

关键词: 视黄酸, 神经管畸形, 基因表达, 趋势分析

Abstract:

Objective ·To explore the molecular regulatory mechanism of neural tube defect (NTD) induced by retinoic acid (RA) in mouse embryos, and reveal the gene expression regularity of neural tube closure in mice. Methods ·Based on the high-quality brain vesicle transcriptome data of mouse embryo during the critical period of neural tube closure [embryonic day 8.5 (E8.5), E9.5 and E10.5], the gene expression trend data of the NTD group and the control group were obtained by using Short Time-series Expression Miner (STEM) software. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for genes with different expression trends between the NTD group and the control group. Some candidate genes were screened for validation. Pregnant mice were divided into the NTD group and control group, with 9 mice in each group. Pregnant mice in the NTD group were treated with RA and those in the control group were treated with sesame oil by gavage at E7.5. Foetal rat brain vesicle tissues were collected at E8.5, E9.5 and E10.5 for experiments. Based on the above animal tissues, the screened candidate genes were validated by quantitative real-time PCR (RT-PCR). Results ·A total of 18 255 genes were detected in the transcriptome data of the control group, and the expression patterns of these genes could be summarized into 7 significant profiles. A total of 19 037 gene expression data were detected in the transcriptome data of the NTD group, and gene expression patterns could be summarized into 6 profiles with significant significance. A total of 46 genes in the control group showed an upward trend but a downward trend in the NTD group. They were enriched in the positive and negative regulation of organ development, neuronal apoptosis, oligodendrocyte proliferation, and fibroblast growth factor signaling pathway at the biological process level. At the cellular component level, they were mainly involved in the basic structure of cells and neurons; At the molecular functional level, they were mainly related to the binding of fibroblast growth factor receptor. A total of 61 genes showed a downward trend in the control group but an upward trend in the NTD group. These genes were enriched in functions such as cell lysis and amino acid/ion transport at the biological process level. At the cellular component level, they were enriched in intracellular molecules, particles, extracellular region, intercellular space, etc. At the molecular function level, they were related to the activity of a series of enzymes and transporters. The results of RT-qPCR showed that the transcriptome sequencing data were authentic and reliable. Conclusion ·RA intervention causes abnormal cellular activities and stress responses during mouse embryo development, leading to abnormal embryo development, activation of signalling pathways related to organismal self-protection, and suppression of genes that maintain normal embryo development.

Key words: retinoic acid, neural tube defect, gene expression, trend analysis

中图分类号:

R322.81

曹睿, 卫凯鑫, 张晓娜, 刘雨榕, 张丽. 视黄酸诱导的小鼠神经管畸形基因表达趋势分析[J]. 上海交通大学学报（医学版）, 2024, 44(7): 859-870.

CAO Rui, WEI Kaixin, ZHANG Xiaona, LIU Yurong, ZHANG Li. Trend analysis of differentially expressed genes in retinoic acid-induced neural tube defects in mouse model[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(7): 859-870.

图/表 7

表1 RT-qPCR引物序列

Tab 1 RT-qPCR primer sequences

Gene name	Gene ID	Primer type	Primer sequence (5′→3′)
Vax1	22326	Forward	AAGAAGGACCAGGGCAAGGACTC
Vax1	22326	Reverse	GCACAGGGCGGCAGCAAG
Foxf2	14238	Forward	CGCCACAACCTCTCGCTCAAC
Foxf2	14238	Reverse	GCGGAACGAACCCTCCTCAAAC
Ptprz1	19283	Forward	GTCACCACCACCAAGTATGCTACC
Ptprz1	19283	Reverse	AAACTGCCCACGCTTTAGATCCTG
Stc2	20856	Forward	GCAGGAAGTGTCCAGCAATTAGG
Stc2	20856	Reverse	GCAGGTTCACAAGGTCCACATAG
Slc7a3	11989	Forward	TGTCCATTTGTGTTCTCATCCTCAG
Slc7a3	11989	Reverse	GCTTCTCTGCTTCAAGTGTCTCC
Fgf1	14164	Forward	TTATACGGCTCGCAGACACC
Fgf1	14164	Reverse	TCTGGCCATAGTGAGTCCGA

图1 正常组和处理组小鼠胚胎在E8.5、E9.5和E10.5的发育情况

Fig 1 Development of mouse embryos in the control group and the NTD group at E8.5, E9.5 and E10.5

图2 正常组具有显著意义的基因表达趋势结果Note： The color lines in each box represent the expression trend of all genes in the profile at different time points. Given a time series vector of gene expression values (v0, v1, v2, .vi.., vn), log2(vi/v0) is the log2 value of the ratio of the expression level of a certain gene at the i-th time point to the expression level at the 0-th time point. FDR—false discovery rate.

Fig 2 Significant gene expression patterns in the control group

图3 处理组具有显著意义的基因表达趋势结果Note： The color lines in each box represent the expression trend of all genes in the profile at different time points. Given a time series vector of gene expression values (v0, v1, v2, .vi.., vn), log2(vi/v0) is the log2 value of the ratio of the expression level of a certain gene at the i-th time point to the expression level at the 0-th time point. FDR—false discovery rate.

Fig 3 Significant gene expression patterns in the NTD group

图4 正常组与处理组具有差异表达趋势的基因集概览Note： A. The intersection of genes expressed in the control group with an upward trend and the NTD group with a downward trend. B. The intersection of genes expressed in the control group with a downward trend and NTD group with an upward trend.

Fig 4 Overview of gene sets with differential expression trend between the control group and the NTD group

图5 正常组与处理组表达趋势具有差异的基因的功能富集结果Note： A/B. GO functional enrichment results of genes up-regulated (A) or down-regulated (B) after RA treatment. C/D. KEGG functional enrichment results of genes up-regulated (C) or down-regulated (D) after RA treatment.

Fig 5 Enrichment results of gene function with different expression trend between the control group and the NTD group

图6 RT-qPCR检测候选基因在正常组和处理组E8.5，E9.5和E10.5时的表达水平Note： A. Vax1 mRNA level. B. Foxf2 mRNA level. C. Ptprz1 mRNA level. D. Stc2 mRNA level. E. Slc7a3 mRNA level. F. Fgf1 mRNA level. ①P=0.002, ②P=0.004, ③P=0.029, compared with the NTD group at E8.5; ④P=0.000, ⑤P=0.002, compared with the NTD group at E9.5; ⑥P=0.000, ⑦P=0.005, compared with the NTD group at E10.5.

Fig 6 Expression levels of candidate genes detected by RT-qqPCR in the control group and the NTD group at E8.5, E9.5 and E10.5

参考文献 32

1	ISAKOVIĆ J, ŠIMUNIĆ I, JAGEČIĆ D, et al. Overview of neural tube defects: gene-environment interactions, preventative approaches and future perspectives[J]. Biomedicines, 2022, 10(5): 965.
2	KANCHERLA V. Neural tube defects: a review of global prevalence, causes, and primary prevention[J]. Childs Nerv Syst, 2023, 39(7): 1703-1710.
3	RAYON T, STAMATAKI D, PEREZ-CARRASCO R, et al. Species-specific pace of development is associated with differences in protein stability[J]. Science, 2020, 369(6510): eaba7667.
4	ZAGANJOR I, SEKKARIE A, TSANG B L, et al. Describing the prevalence of neural tube defects worldwide: a systematic literature review[J]. PLoS One, 2016, 11(4): e0151586.
5	KANG L Y, GUO Z R, SHANG W J, et al. Perinatal prevalence of birth defects in the Mainland of China, 2000—2021: a systematic review and meta-analysis[J]. World J Pediatr, 2024. DOI: 10.1007/s12519-023-00786-8.
6	MOORE C A, LI S, LI Z, et al. Elevated rates of severe neural tube defects in a high-prevalence area in northern China[J]. Am J Med Genet, 1997, 73(2): 113-118.
7	LI Z W, REN A G, ZHANG L, et al. Extremely high prevalence of neural tube defects in a 4-county area in Shanxi Province, China[J]. Birth Defects Res A Clin Mol Teratol, 2006, 76(4): 237-240.
8	VAN GOOL J D, HIRCHE H, LAX H, et al. Folic acid and primary prevention of neural tube defects: a review[J]. Reprod Toxicol, 2018, 80: 73-84.
9	YU J, WANG L, PEI P, et al. Reduced H3K27me3 leads to abnormal Hox gene expression in neural tube defects[J]. Epigenetics Chromatin, 2019, 12(1): 76.
10	ERNST J, BAR-JOSEPH Z. STEM: a tool for the analysis of short time series gene expression data[J]. BMC Bioinformatics, 2006, 7: 191.
11	CAO R, LI J Q, ZHANG L, et al. Analysis of genes associated with both neural tube defects and neuroectodermal tumors[J]. Med Sci Monit, 2022, 28: e936079.
12	SUN Y Q, ZHANG J, WANG Y F, et al. miR-222-3p is involved in neural tube closure by directly targeting Ddit4 in RA induced NTDs mouse model[J]. Cell Cycle, 2021, 20(22): 2372-2386.
13	CHAI Z, YANG L H, YU B F, et al. p38 mitogen-activated protein kinase-dependent regulation of SRC-3 and involvement in retinoic acid receptor α signaling in embryonic cortical neurons[J]. IUBMB Life, 2009, 61(6): 670-678.
14	LIU J G, ZHOU R, HE Q R, et al. Calmodulin kinase Ⅱ activation of mitogen-activated protein kinase in PC12 cell following all-trans retinoic acid treatment[J]. Neurotoxicology, 2009, 30(4): 599-604.
15	EAGLESON G, FERREIRO B, HARRIS W A. Fate of the anterior neural ridge and the morphogenesis of the Xenopus forebrain[J]. J Neurobiol, 1995, 28(2): 146-158.
16	HALLONET M, HOLLEMANN T, WEHR R, et al. Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain[J]. Development, 1998, 125(14): 2599-2610.
17	PENG L, NIU Z M, CHEN J P, et al. Association of genetic polymorphisms of VAX1, MAFB, and NTN1 with nonsyndromic cleft lip with or without cleft palate in Chinese population[J]. Mol Genet Genomics, 2022, 297(2): 553-559.
18	HE W H, KANG Y B, ZHU W, et al. FOXF2 acts as a crucial molecule in tumours and embryonic development[J]. Cell Death Dis, 2020, 11(6): 424.
19	EVERSON J L, FINK D M, YOON J W, et al. Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis[J]. Development, 2017, 144(11): 2082-2091.
20	WU Q, LI W, YOU C G. The regulatory roles and mechanisms of the transcription factor FOXF2 in human diseases[J]. PeerJ, 2021, 9: e10845.
21	XIA Z, OUYANG D, LI Q, et al. The expression, functions, interactions and prognostic values of PTPRZ1: a review and bioinformatic analysis[J]. J Cancer, 2019, 10(7): 1663-1674.
22	KUBOYAMA K, FUJIKAWA A, SUZUKI R, et al. Role of chondroitin sulfate (CS) modification in the regulation of protein-tyrosine phosphatase receptor type Z (PTPRZ) activity: pleiotrophin-PTPRZ-A signaling is involved in oligodendrocyte differentiation[J]. J Biol Chem, 2016, 291(35): 18117-18128.
23	KLAUSMEYER A, GARWOOD J, FAISSNER A. Differential expression of phosphacan/RPTPβ isoforms in the developing mouse visual system[J]. J Comp Neurol, 2007, 504(6): 659-679.
24	ROLL L, LESSMANN K, BRÜSTLE O, et al. Cerebral organoids maintain the expression of neural stem cell-associated glycoepitopes and extracellular matrix[J]. Cells, 2022, 11(5): 760.
25	PASTOR M, FERNÁNDEZ-CALLE R, GERONIMO B D, et al. Development of inhibitors of receptor protein tyrosine phosphatase β/ζ (PTPRZ1) as candidates for CNS disorders[J]. Eur J Med Chem, 2018, 144: 318-329.
26	QIE S, SANG N. Stanniocalcin 2 (STC2): a universal tumour biomarker and a potential therapeutical target[J]. J Exp Clin Cancer Res, 2022, 41(1): 161.
27	JEON Y, SHIN J E, KWON M, et al. In vivo gene delivery of STC2 promotes axon regeneration in sciatic nerves[J]. Mol Neurobiol, 2021, 58(2): 750-760.
28	CAO Y Z, JIA Q H, XING Y X, et al. STC2 inhibits hepatic lipid synthesis and correlates with intramuscular fatty acid composition, body weight and carcass traits in chickens[J]. Animals, 2024, 14(3): 383.
29	GAO H J, WU G Y, SPENCER T E, et al. Select nutrients in the ovine uterine lumen. Ⅲ. Cationic amino acid transporters in the ovine uterus and peri-implantation conceptuses[J]. Biol Reprod, 2009, 80(3): 602-609.
30	KIM J J, KHALID O, NAMAZI A, et al. Discovery of consensus gene signature and intermodular connectivity defining self-renewal of human embryonic stem cells[J]. Stem Cells, 2014, 32(6): 1468-1479.
31	KOKKONEN H, SIREN A, MÄÄTTÄ T, et al. Identification of microduplications at Xp21.2 and Xq13.1 in neurodevelopmental disorders[J]. Mol Genet Genomic Med, 2021, 9(12): e1703.
32	NURCOMBE V, FORD M D, WILDSCHUT J A, et al. Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan[J]. Science, 1993, 260(5104): 103-106.

[1]	孙思远, 刘媛琪, 崔怡雯, 黄紫晗, 梅李, 代庆刚, 江凌勇. 成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证[J]. 上海交通大学学报（医学版）, 2024, 44(6): 676-686.
[2]	克德尔亚·艾山江, 傅怡, 赖冬林, 邬海龙, 龚伟. 肝细胞癌相关的核编码线粒体基因及临床信息的综合预后模型[J]. 上海交通大学学报（医学版）, 2024, 44(1): 1-12.
[3]	侯宗良, 杨琴, 李少白, 雷鸣. 癌睾丸基因的基因表达程序分析[J]. 上海交通大学学报（医学版）, 2023, 43(8): 945-954.
[4]	韩夏夏, 蒋扬, 顾霜霜, 戴黛, 沈南. 系统性红斑狼疮患者免疫细胞代谢通路转录组分析[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1197-1207.
[5]	王建茹, 彭广操, 朱明军. 基于GEO数据库和生物信息学分析筛选小鼠心肌缺血再灌注损伤相关的潜在枢纽基因[J]. 上海交通大学学报(医学版), 2022, 42(1): 51-62.
[6]	李雨晨, 白丽莲, 黄荷凤, 刘欣梅. 人胸腺基质发育及退化的单细胞转录组分析[J]. 上海交通大学学报(医学版), 2021, 41(7): 865-875.
[7]	王昱欢, 丁奕岑, 蔡瑶雨, 康亚妮. 差异表达微RNA作为多囊卵巢综合征生物标志物的研究[J]. 上海交通大学学报(医学版), 2021, 41(11): 1429-1435.
[8]	李超，糜坚青，王瑾. 费城染色体样急性淋巴细胞白血病的研究进展[J]. 上海交通大学学报(医学版), 2020, 40(9): 1294-1301.
[9]	建方方1，车晓霞2，冯炜炜1. 子宫内膜癌中长链非编码RNA表达谱的生物信息学分析[J]. 上海交通大学学报(医学版), 2020, 40(6): 768-775.
[10]	朱屿倩，吴凌云. m6A甲基化修饰在血液系统恶性肿瘤中的作用研究进展[J]. 上海交通大学学报（医学版）, 2020, 40(3): 396-.
[11]	程影 1，鞠培俊 1，王卫娣 1，方钰 1，林关宁 1, 2，崔东红 1. 精神分裂症易感基因在人类与小鼠脑组织单细胞中的表达差异分析[J]. 上海交通大学学报（医学版）, 2019, 39(2): 113-.
[12]	刘祖银，李清，陈丽君，等. 全反式视黄酸通过RARγ蛋白直接调控PPARγ2蛋白抑制骨髓间充质干细胞成脂分化[J]. 上海交通大学学报（医学版）, 2015, 35(5): 682-.
[13]	陈丽君，任兰，刘祖银，等. 孕期边缘型维生素A缺乏对新生鼠RARα、Src和NMDAR1表达的影响[J]. 上海交通大学学报（医学版）, 2015, 35(4): 500-.
[14]	黄宝星, 孙福康. 原发性醛固酮增多症的分子机制研究进展[J]. , 2013, 33(5): 685-.
[15]	王洁, 阿吉艾克拜尔·艾萨, 马海蓉, 等. GPER：雌激素相关疾病治疗的新靶点[J]. , 2013, 33(2): 225-.

视黄酸诱导的小鼠神经管畸形基因表达趋势分析

Trend analysis of differentially expressed genes in retinoic acid-induced neural tube defects in mouse model

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 32

相关文章 15

编辑推荐

Metrics